Phytanic acid induced neurological alterations in rat brain synaptosomes and its attenuation by melatonin.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) (Phyt) is a saturated branched chain fatty acid which originates after the breakdown of chlorophyll molecule, phytol. It plays an important role in a variety of metabolic disorders with peroxisomal impairments. The aim of our investigation was to evaluate the adverse effects of Phyt on synaptic functions by using synaptosomal preparation of rat brain as an in vitro model and the possible protective role of melatonin against Phyt-induced neurotoxicity. Melatonin is an antioxidant, secreted by the pineal gland. Melatonin and its metabolites have neuroprotective effects on cellular stress, by reducing reactive oxygen species (ROS) and reactive nitrogen species (RNS). In the present investigation, synaptosomes prepared from rat brain were co-treated with melatonin (10µM) and Phyt (50µM) for 2h. Co-treatment of Phyt with melatonin significantly restored the altered levels of protein carbonyl (PC) contents and lipid peroxidation (LPO). It also replenished the Phyt-induced alterations on the levels of non-enzymatic antioxidant defence reduced glutathione (GSH), enzymatic antioxidants such as catalase (CAT) and superoxide dismutase (SOD) and synaptosomal integral enzymes such as AChE, Na+, K+-ATPase and MAO. We observed that Phyt induced oxidative stress in synaptosomes as indicated by an elevation in the generation of ROS and melatonin was able to inhibit the elevated ROS generation. Moreover, the neurotoxic effects elicited by Phyt on NO level and membrane potential were totally prevented by the treatment of melatonin. The results of our investigation emphasize the potential use of melatonin as a nutraceutical and mitigatory agent against Phyt-induced oxidative stress.

Phytanic Acid-Induced Neurotoxicological Manifestations and Apoptosis Ameliorated by Mitochondria-Mediated Actions of Melatonin.

Phytanic acid, a saturated branched chain fatty acid and a major constituent of human diet, is predominantly found in dairy products, meat, and fish. It is a degradation product from the phytol side chain of chlorophyll. Degradation of PA is known to occur mainly in peroxisomes via α-oxidation and in mitochondria via ß-oxidation. Due to its ß-methyl group present at the 3-position of the carbon atoms, PA cannot be ß-oxidized. Although alteration in the metabolism of PA may play an important role in neurodegeneration, the exact mechanism behind it remains to be evaluated. In this study, we have described the potential of PA to induce neurotoxicity as an in vitro model (neuronal cell line, SH-SY5Y cells). Cells were pretreated with melatonin (10 µM) for 1 h followed by with and without PA (100 µM) for 24 h. In the present study, our data has confirmed that PA markedly increased both intracellular reactive oxygen species and reactive nitrogen species levels. Our results have shown that PA treatment did not induce cell death by cleavage of caspase-3/PARP-1 mediated by mitochondria through intrinsic pathways; however, PA induced nitric oxide-dependent apoptosis in SH-SY5Y cells. Additionally, melatonin pretreatment reduced the cell death in SH-SY5Y cells. Melatonin also effectively exerted an antiapoptotic and anti-inflammatory action by regulating Bax, Bcl-2, p-NFκB, and iNOS expressions in SH-SY5Y cells. These results suggested that melatonin acted as an antioxidative and antiapoptotic agent by modulating ROS, apoptotic proteins, and inflammatory responses under BCFA-induced neurotoxic conditions. The protective effects of melatonin depend on direct scavenging activity of free radicals and indirect antioxidant effects. Further deciphering of the cellular and molecular mechanism associated with neuroprotection by melatonin is warranted in BCFA-induced neurotoxicity.

Phytanic acid induces Neuro2a cell death via histone deacetylase activation and mitochondrial dysfunction.

Phytanic acid is a branched fatty acid that is a metabolic intermediate of chlorophyll. In this study, the effects of phytanic acid on Histone deacetylase (Hdac) activity were examined in an in vitro enzyme assay and in neuronal Neuro2a cells. Several fatty acids have been shown to be Hdac inhibitors, but phytanic acid enhanced the enzyme activity in vitro. In Neuro2a cells, phytanic acid significantly reduced histone acetylation and induced cell death, which was inhibited by an Hdac inhibitor, sodium butyrate. Theophylline, a common Hdac activator, had a similar effect on Neuro2a cell viability, and this effect was also inhibited by sodium butyrate. Phytanic acid decreased the level of intracellular active mitochondria, while butyrate increased this level. The cytotoxic effect of phytanic acid was also abolished by a caspase-9 inhibitor. Apicidin, a Hdac2- and 3-specific inhibitor, reduced the cellular toxicity, which suggests that the toxicity of phytanic acid depends on activation of the Hdac2 and 3 subtypes. Overall, these results show that phytanic acid induces mitochondrial abnormality and cell death via activation of Hdac2, 3 in Neuro2a cells. This effect of Hdac activation by phytanic acid may produce neuronal damage in Refsum disease and other peroxisomal disorders, which is caused by accumulation of phytanic acid.

Toxicological issues associated with production and processing of meat.

Meat is a very complex and continuously changing ex vivo system of various high- and low-molecular substances that can be used for satisfying needs of the human organism for metabolic energy, building material and fulfilling of the other vital functions. A great majority of these substances are useful and safe for the consumer. Yet, meat and meat products may always contain substances exerting detrimental effects to the consumer's organism. The present paper is a literature review of the most important potentially toxic substances found in meat and meat products; their classification, ways of getting into the meat or formation during meat processing, undesirable physiological outcomes and biochemical mechanisms of their toxic effects, and methods for reduction of these responses.

Phytanic acid and the risk of non-Hodgkin lymphoma.

Greater consumption of red meat, processed meat and dairy products has been associated with an increased risk of non-Hodgkin lymphoma (NHL) in several previous reports. Phytanic acid, a saturated fatty acid obtained primarily through the consumption of ruminant meat and dairy products, may offer a potential underlying mechanism for these associations. In a population-based case-control study of 336 cases and 460 controls conducted in Nebraska during 1999-2002, we examined whether phytanic acid-containing foods or total phytanic acid intake, estimated from a food frequency questionnaire and the published phytanic acid values of 151 food items, were associated with increased NHL risk. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals for overall NHL and the common NHL histologic subtypes. In multivariable models, higher intakes of density-adjusted beef [OR(T3 vs. T1) = 1.5 (1.1-2.2); P(trend) = 0.02], total dairy products [OR = 1.5 (1.1-2.2); P(trend) = 0.02) and milk [OR = 1.6 (1.1-2.3); P(trend) = 0.01] were associated with an increased risk of NHL. Intake of total phytanic acid was positively associated with NHL risk [OR = 1.5 (1.0-2.1); P(trend) = 0.04]. In analyses stratified by NHL subtype, greater consumption of beef was associated with an increased risk of diffuse large B-cell lymphoma, and greater consumption of milk was associated with an increased risk of follicular lymphoma (FL). Total phytanic acid intake was associated with an increased risk of FL and small lymphocytic lymphoma/chronic lymphocytic leukemia. Our results provide support that total phytanic acid and phytanic acid-containing foods may increase NHL risk.

Neurochemical evidence that phytanic acid induces oxidative damage and reduces the antioxidant defenses in cerebellum and cerebral cortex of rats.

AIMS: In the present work we investigated the in vitro effects of phytanic acid (Phyt), that accumulates in Refsum disease and other peroxisomal diseases, on important parameters of oxidative stress in cerebellum and cerebral cortex from young rats. MAIN METHODS: The parameters thiobarbituric acid-reactive substances levels (TBA-RS; lipid peroxidation), carbonyl formation and sulfhydryl oxidation (protein oxidative damage) and the concentrations of the most important nonenzymatic antioxidant defense reduced glutathione (GSH) were determined. KEY FINDINGS: It was observed that Phyt significantly increased TBA-RS levels in both cerebral structures. This effect was prevented by the antioxidants alpha-tocopherol and melatonin, suggesting the involvement of free radicals. Phyt also provoked protein oxidative damage in both cerebellum and cerebral cortex, as determined by increased carbonyl content and sulfhydryl oxidation. Furthermore, Phyt significantly diminished the concentrations of GSH, while melatonin and alpha-tocopherol treatment totally blocked this effect. We also verified that Phyt does not behave as a direct acting oxidant, since Phyt did not oxidize commercial solutions of GSH and reduced cytochrome c to Phyt in a free cell medium. SIGNIFICANCE: Our data indicate that oxidative stress is elicited in vitro by Phyt, a mechanism that may contribute at least in part to the pathophysiology of Refsum disease and other peroxisomal disorders where Phyt is accumulated.

A study of the cytotoxicity of branched-chain phytanic acid with mitochondria and rat brain astrocytes.

Phytanic acid, a saturated fatty acid of 20-carbon-atoms with isoprenoic structure, is formed from the phytol-side chain of chlorophyll in ruminants. Degradation of phytanic acid is blocked in Refsum disease by several enzymatic defects of peroxisomal degradation of branched-chain fatty acids. Refsum disease is an inherited neurological disorder progressively developing from early childhood to adultness. Clinical signs are attributed to toxicity of phytanic acid, which accumulates to unusually high levels in the tissue and serum of patients suffering from untreated Refsum disease. We report here that hippocampal astrocytes isolated from rat brain, which were exposed to phytanic acid (50 microM) die within a few hours. In situ depolarization of mitochondria and an increase of cytosolic Ca2+ precede cell death. Therefore, we also investigated the influence of phytanic acid on physiology of mitochondria isolated from rat brain. Mitochondria become functionally impaired by phytanic acid, as indicated by uncoupling (resting state), inhibition of the electron transport (state 3), stimulation of ROS-generation, decline of Ca2+ loading and severe release of cytochrome c. Thus, phytanic acid seems to initiate astrocyte cell death by activating the mitochondrial route of apoptosis.

Mechanism of toxicity of the branched-chain fatty acid phytanic acid, a marker of Refsum disease, in astrocytes involves mitochondrial impairment.

Phytanic acid is a saturated branched-chain fatty acid, which is formed by bacterial degradation of chlorophyll in the intestinal tract of ruminants. The methyl group in beta-position prevents degradation of phytanic acid by the beta-oxidation pathway. Therefore, degradation of phytanic acid is initiated by alpha-oxidation in peroxisomes. The inherited peroxisomal disorder Refsum disease is characterised by accumulation of phytanic acid. Unusually high concentrations of phytanic acid can be found in the plasma of Refsum disease patients, who suffer from neurodegeneration and muscle dystrophy. Phytanic acid has been suggested to be causally involved in the clinical symptoms. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid in rat hippocampal astrocytes by monitoring the cytosolic Ca(2+) concentration, the mitochondrial membrane potential (Deltapsi(m)), the generation of reactive oxygen species as well as the cellular ATP level. In response to phytanic acid (100 microM) cytosolic Ca(2+) was quickly increased. The phytanic acid-evoked Ca(2+) response was transient and involved activation of intracellular Ca(2+) stores. In isolated rat brain mitochondria, phytanic acid dissipated Deltapsi(m) in a reversible and dose-dependent manner. Moreover, phytanic acid released cytochrome c from mitochondria. Depending on the mitochondrial activity state, phytanic acid either stimulated or inhibited the electron flux within the respiratory chain. In addition, phytanic acid induced substantial generation of reactive oxygen species in isolated mitochondria as well as in intact cells. Phytanic acid caused cell death of astrocytes within a few hours of exposure. In conclusion, we suggest that phytanic acid initiates astrocyte cell death by activating the mitochondrial route of apoptosis.

The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes.

The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.

Potentiation of the teratogenic effects induced by coadministration of retinoic acid or phytanic acid/phytol with synthetic retinoid receptor ligands.

Previous studies in our laboratory identified retinoid-induced defects that are mediated by RAR-RXR heterodimerization using interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in mice (Elmazar et al. 1997, Toxicol Appl Pharmacol 146:21-28; Elmazar et al. 2001, Toxicol Appl Pharmacol 170:2-9; Nau and Elmazar 1999, Handbook of experimental pharmacology, vol 139, Retinoids, Springer-Verlag, pp 465-487). The present study was designed to investigate whether these RAR-RXR heterodimer-mediated defects can be also induced by interactions of natural and synthetic ligands for retinoid receptors. A non-teratogenic dose of the natural RXR agonist phytanic acid (100 mg/kg orally) or its precursor phytol (500 mg/kg orally) was coadministered with a synthetic RARalpha-agonist (Am580; 5 mg/kg orally) to NMRI mice on day 8.25 of gestation (GD8.25). Furthermore, a non-teratogenic dose of the synthetic RXR agonist LGD1069 (20 mg/kg orally) was also coadministered with the natural RAR agonist, all- trans-retinoic acid (atRA, 20 mg/kg orally) or its precursor retinol (ROH, 50 mg/kg orally) to NMRI mice on GD8.25. The teratogenic outcome was scored in day-18 fetuses. The incidence of Am580-induced resorptions, spina bifida aperta, micrognathia, anotia, kidney hypoplasia, dilated bladder, undescended testis, atresia ani, short and absent tail, fused ribs and fetal weight retardation were potentiated by coadministration of phytanic acid or its precursor phytol. Am580-induced exencephaly and cleft palate, which were not potentiated by coadministration with the synthetic RXR agonists, were also not potentiated by coadministration with either phytanic acid or its precursor phytol. LGD1069 potentiated atRA- and ROH-induced resorption, exencephaly, spina bifida, aperta, ear anotia and microtia, macroglossia, kidney hypoplasia, undescended testis, atresia ani, tail defects and fetal weight retardation, but not cleft palate. These results suggest that synergistic teratogenesis can be induced by coadministration of a natural RXR ligand (phytanic acid) with a synthetic RAR agonist (Am580). Thus, certain potentially useful therapeutic agents or nutritional factors such as phytanic acid should be tested for teratogenic risk by coadministration with other retinoid receptor agonists.

Expression of fatty acid binding proteins inhibits lipid accumulation and alters toxicity in L cell fibroblasts.

High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. Although fatty acid binding proteins (FABPs) are thought to be protective, this hypothesis has not previously been examined. Phytanic acid (branched chain, 16-carbon backbone) induced lipid accumulation in L cell fibroblasts similar to that observed with palmitic acid (unbranched, C(16)): triacylglycerol >> free fatty acid > cholesterol > cholesteryl ester >> phospholipid. Although expression of sterol carrier protein (SCP)-2, SCP-x, or liver FABP (L-FABP) in transfected L cells reduced [(3)H]phytanic acid uptake (57-87%) and lipid accumulation (21-27%), nevertheless [(3)H]phytanic acid oxidation was inhibited (74-100%) and phytanic acid toxicity was enhanced in the order L-FABP >> SCP-x > SCP-2. These effects differed markedly from those of [(3)H]palmitic acid, whose uptake, oxidation, and induction of lipid accumulation were not reduced by L-FABP, SCP-2, or SCP-x expression. Furthermore, these proteins did not enhance the cytotoxicity of palmitic acid. In summary, intracellular FABPs reduce lipid accumulation induced by high levels of branched-chain but not straight-chain saturated fatty acids. These beneficial effects were offset by inhibition of branched-chain fatty acid oxidation that correlated with the enhanced toxicity of high levels of branched-chain fatty acid.

Prevention of vitamin A teratogenesis by phytol or phytanic acid results from reduced metabolism of retinol to the teratogenic metabolite, all-trans-retinoic acid.

Previous studies in our laboratory showed a synergistic interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in regard to teratogenic effects produced in mice (M. M. Elmazar et al., 2001, TOXICOL: Appl. Pharmacol. 170, 2-9). In the present study the influence of phytol and phytanic acid (a RXR-selective ligand) on the teratogenicity of retinol and the RAR-selective ligand all-trans-retinoic acid was investigated by coadministration experiments on day 8.25 of gestation in NMRI mice. Phytol and phytanic acid, noneffective when administered alone, did not potentiate the teratogenicity induced by retinol or all-trans-retinoic acid. On the contrary, phytol and phytanic acid greatly reduced retinol-induced teratogenic effects (ear anotia, tail defects, exencephaly). The effect of phytol on all-trans-retinoic acid teratogenesis was limited (only resorptions and tail defects were reduced). Pharmacokinetic studies in nonpregnant animals revealed that phytol coadministration with retinol reduced plasma levels of retinol and retinyl esters, and drastically reduced the levels of the teratogenic retinol metabolite, all-trans-retinoic acid. Phytanic acid also reduced the oxidative metabolism and teratogenic effects of retinol. These results indicate that phytol and phytanic acid did not synergize with retinol and all-trans-retinoic acid in our mouse teratogenesis model. Instead, phytol and phytanic acid effectively blocked the teratogenic effects of retinol by drastically reducing the metabolic production of all-trans-retinoic acid. Phytol and phytanic acid may be useful for the prevention of vitamin A teratogenicity.